DESCRIPTION (Adapted from applicants' abstract) Evidence is emerging that retinoid signaling plays a pivotal role in cardiac development and metabolism. Both vitamin A deficiency and excess during development lead to structural abnormalities of the heart. Moreover, targeted ablation of the retinoid X receptor a (RXRa), a class II member of the nuclear receptor transcription factor (NRTF) family, leads to cardiac defects and embryonic lethality in mice. To identify proteins which interact with RXRa, the yeast two-hybrid system was used to screen a heart cDNA library with RXRa. A cardiac-enriched RXR associated protein, RAP 2 was identified. Based on sequence homology, RAP 2 is a novel member of the immunophilin family. Others have shown that ligand-mediated nuclear translocation of the class I NRTFs (e.g., glucocorticoid receptor) requires the immunophilin FKBP59; the applicant's preliminary data indicate that RAP 2 contains the structural domains of FKBP59. This application is designed to test the hypothesis that RAP 2 plays a critical role in intracellular trafficking of RXRa and that the biological effects of retinoids in the heart are modulated by intracellular trafficking events. The aims of the study are: I) Characterization of the RXRa/RAP 2 interaction and its specificity using wild-type and mutant proteins in protein:protein interaction studies including GST pulldown assays, co-immunoprecipitations, DNA:protein:protein interaction studies and the yeast two-hybrid interaction assay. II) Delineation of other molecules involved in RAP 2-mediated intracellular trafficking of RXR by characterization of the putative heterocomplex of proteins involved in the RAP 2/RXR interaction, characterization of the FK506 binding properties of RAP 2 and by cellular immunolocalization studies. III) Delineation of the role of RAP 2 in the retinoid signaling pathway using co-transfection studies with a retinoid responsive reporter and in a retinoid-dependent in vitro cell differentiation system using mutants as characterized above. The long term goals of this project are to understand the genesis of cardiac malformations and the role of retinoid signaling in this process. Dr. Clabby will be supervised during the period of support by Dr. Daniel Kelly, director of the Center for Cardiovascular Research. The funds provided by this award will facilitate Dr. Clabby's development into an independent scientist.